In this project proposal, the folding and unfolding reactions of the model system recombinant alpha-macrophage colony stimulating factor beta (rhm-CSFbeta) will be characterized. Stopped flow CD will be used to analyze the kinetics of the formation of the first detectable kinetic intermediate on the refolding pathway (monomeric rhm-CSFbeta with reformed secondary structure) and the effects of carboxamido-methylation of thiols on the regaining of secondary structure. HPLC and fluorescence intensity changes will be used to examine the refolding process in the slow time domain (minutes-hours) where the effect of parameters such as protein concentration on the refolding pathway will be examined. The time- dependence of deuterium/hydrogen exchanges kinetics for discrete rhm- CSFbeta domains during unfolding will be determined by electrospray mass spectrometry on peptic peptides generated after rapid-mix-quench exchange experiments. These experiments should permit mapping of the kinetic pathway of rhm-MCSFbeta folding and a detailed analysis of the first detectable kinetic step in the pathway as well as a correlation between solvent accessible protein domains and the formation/decomposition of kinetic intermediates. The effects of chemical insult due to alkylation and oxidation of thiols and oxidation of methionine on the energetics on folding kinetics will be analyzed and correlated with the site of modifications.